Understanding Early Post-Mortem Biochemical Processes Underlying

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Understanding early post-mortem biochemical processes underlying meat colour and pH decline in the Longissimus thoracis muscle of young Blond d’Aquitaine bulls using protein biomarkers Mohammed Gagaoua, Claudia EM Terlouw, Didier Micol, Abdelghani Boudjellal, Jean-François Hocquette, and Brigitte Picard J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02615 • Publication Date (Web): 09 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Agricultural and Food Chemistry

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Understanding early post-mortem biochemical processes underlying meat colour and pH

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decline in the Longissimus thoracis muscle of young Blond d’Aquitaine bulls using

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protein biomarkers

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Mohammed Gagaoua1,2,3, E. M. Claudia Terlouw1,2, Didier Micol1,2, Abdelghani Boudjellal3,

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Jean-François Hocquette1,2, Brigitte Picard 1,2 (*)

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INRA, UMR 1213 Herbivores, F-63122 Saint-Genès Champanelle, France Clermont Université, VetAgro Sup, UMR 1213 Herbivores, B.P. 10448, F-63000 ClermontFerrand, France 3 Equipe Maquav, INATAA, Université Frères Mentouri Constantine, Route de Ain El-Bey, 25000, Constantine, Algeria 2

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* Corresponding author: Brigitte Picard, Tel: 0033473624056, Fax: 0033473624639,

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Email: [email protected]

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ABSTRACT

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Many studies on colour biochemistry and protein biomarkers were undertaken in postmortem beef muscles after 24 or more hours. The present study was conducted on Longissimus thoracis muscles of 21 Blond d’Aquitaine young bulls to evaluate the relationships between protein biomarkers present during the early post-mortem and known to be related to tenderness with pH decline and colour development. pH at 45 min, 3h and 30h pm were correlated with 3, 7 and 6 biomarkers, respectively. L* a* b* colour coordinates 24h p-m were correlated with 9, 5 and 8 protein biomarkers, respectively. Regression models included Hsp proteins and explained between 47 and 59% of the variability between individuals in pH and between 47 and 65% of the variability in L* a* b* colour coordinates. Proteins correlated with pH and/or colour coordinates were involved in apoptosis or had antioxidative or chaperone activities. Main results include the negative correlations between pH45min, pH3h and pHu and Prdx6, which may be explained by the anti-oxidative and phospholipase activities of this biomarker. Similarly, inducible Hsp70-1A/B and µ-calpaïn were correlated with L* a* b* coordinates, due to the protective action of Hsp70-1A/B on the proteolytic activities of µ-calpain on structural proteins. Correlations existed further between MDH1, ENO3 and LDH-B and pH decline and colour stability probably due to the involvement of these enzymes in the glycolytic pathway, and thus, the energy status of the cell. The present results show that research using protein indicators may help to understand early p-m biological mechanisms involved in pH and beef colour development.

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Keywords: Beef; Blond d’Aquitaine; Meat colour; pH; protein biomarkers; post-mortem;

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Biological mechanisms.

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INTRODUCTION

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Meat colour is an organoleptic characteristic with a major influence on purchase decisions

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1, 2

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by consumers

. Colour is influenced by the content and physico-chemical state of

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myoglobin and by the meat structure

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high ultimate pH, muscle fibres are more tightly packed together as a result of increased

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water-holding capacity of muscle protein. As a consequence, its surface will scatter light less

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and the meat will appear darker

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biochemical reactions and structural characteristics of the muscles and may also influence

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colour 5, 6.

3, 4

. The latter is directly related to the ultimate pH: at

3, 4

. The rate of post-mortem pH decline influences

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Myoglobin (Mb) plays further a central role in meat colour. Mb content depends on the

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muscle but also on other factors such as breed, age and diet 1, 2. Slow-twitch oxidative muscle

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fibres are rich in Mb compared to fast-twitch glycolytic fibres 3. Mb contains a haematin

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nucleus with a central iron atom. The iron atom of Mb may be reduced (DeoxyMb; purple),

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oxygenised (MbO2: bright red) or oxidised (MetMb; brown) 7. The interconversion of the

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three forms in fresh beef depends on the levels of antioxidants and pro-oxidants present 8.

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Loss of reducing activity in meat during storage is due to a combination of factors including

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pH decline, depletion of required substrates and co-factors particularly NADH, the presence

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of secondary lipid oxidation products, pH-induced denaturation of the enzymes and ultimately

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the complete loss of structural integrity and functional properties of the mitochondria 1, 8, 9.

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Comparative transcriptomic and proteomic studies have revealed biochemical pathways

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involved in meat sensory qualities, including tenderness, juiciness, water-holding capacity 10-

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13

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resistance, apoptosis or energy metabolism

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be related to meat colour development and stability, particularly those related to oxidative

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processes and the redox status of the muscle, to energy metabolism and consequently to the

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status of the NADH pool and pH decline, and to apoptosis 16-19.

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. These pathways are related to myofibril structure, proteolysis, heat stress, oxidative stress 12, 14, 15

. At least part of these pathways may also

Such transcriptomic and proteomic studies have allowed our laboratory to establish a list of 13

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proteins, known as biomarkers, potentially predictive of certain sensory qualities

. Many

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studies on colour biochemistry and protein biomarkers were undertaken in post-mortem beef

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muscles after 24 or more hours post-mortem. The present study uses the earlier identified

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biomarkers to evaluate the relationships between protein status in the early post-mortem

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period, subsequent pH decline, and beef colour as evaluated by L* a* b* coordinates. The 2 ACS Paragon Plus Environment

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study uses a high through-put technique and was conducted on young bulls of the French

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Blond d’Aquitaine (BA) breed, characterised by good muscle development, producing tender

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meat 20 but with a relatively pale colour. MATERIALS AND METHODS

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Animals and sampling

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This study was part of the European ProSafeBeef project (FOOD-CT-2006-36241).

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Twenty-one Blond d’Aquitaine (BA) young bulls reared under intensive conditions and

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finished over two consecutive years (two replicated groups) were used. At 12 months of age,

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they were subjected to a 105 day finishing period until slaughter. Diets consisted of

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concentrate (75%) and straw (25%). Before slaughter, all animals were food deprived for 24 h

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and had free access to water. The animals at a live weight of 635 ± 52 kg were slaughtered at

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the experimental abattoir of the INRA Research centre in compliance with the current ethical

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guidelines for animal welfare. Bulls were directly transported (4.5 ± 0.1 min) in a lorry (3 x 2

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m livestock compartment) from the experimental farm to the experimental abattoir situated at

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2 km from the rearing building, with 2 bulls of the same home pen per transport to avoid

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social isolation stress. After unloading, they were slaughtered within 3 min 21. The mean hot

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carcass weight was 410 ± 42 kg (range from 337 – 500 kg). The carcasses were not

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electrically stimulated and they were chilled and stored at 4°C from 1 h until 24 h post-

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mortem (p-m). Longissimus thoracis (LT, mixed fast oxido-glycolytic) muscle samples were

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excised from the left side of the 6th rib 30 min p-m and frozen in liquid nitrogen before storage

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at –80°C until protein extractions for Dot-Blot analysis or Myosin Heavy chains (MyHC)

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quantification.

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pH measurements

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For pH45min measurement, biopsies were made from the carcasses (10th rib), while pH3h and

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ultimate pH (30h) were measured directly on the carcass (right side). pH was determined as in

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Bourguet et al.

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immediately homogenized in 18 mL of 5 mM sodium iodoacetate, and stored at 4°C. The pH

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of the homogenate was measured the following day at 6°C. For pH3h and ultimate pH (30h),

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the pH was recorded directly on the carcass between the 6th and 7th rib using a pH meter

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(Hanna Instruments, HI9025) equipped with a glass electrode suitable for meat penetration.

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For each time point, five measurements were made (positioned on a horizontal line with about

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1.5 cm between two measurements).

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. Briefly, 2 g of each LT sample excised 45 min after slaughter were



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Meat colour

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The LT muscle was excised from the stored carcass at the level of the 6th rib thoracis

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vertebra 24 h p-m to determine colour attributes. Instrumental meat colour measurements

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were recorded for lightness (L* = a measure of the light reflected), redness (a* = measures

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positive red and negative green) and yellowness (b* = measures positive yellow and negative

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blue) directly on the tissue of the muscle using a Minolta CR-300 chromameter (Minolta Co.,

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Ltd, Osaka, Japan), with 0° viewing angle, C illuminant and 8 mm measurement aperture.

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Fresh cut slices of muscle of not less than 2.5 cm thick and overwrapped were left on a

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polystyrene tray to refrigerate at 1°C for 1h to allow blooming. Calibration was performed by

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using standard white tiles (Y = 93.58, x = 0.3150 and y = 0.3217) prior to the colour

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determination. Three replicate measurements were taken and an average value was used for

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analysis. Colour coordinates were expressed as L*, a*, b* following the CIE-L*a*b* system.

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Protein extractions

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Total protein extractions were performed to use subsequently the soluble fractions for Dot22

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Blot analysis according to Bouley et al.

. Briefly, 80 mg of muscle was homogenised in a

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denaturation/extraction buffer containing 8.3 M urea, 2 M thiourea, 1% DTT and 2% CHAPS.

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After 30 min of centrifugation at 10 000g at 8 °C, the supernatant was stored at – 20°C until

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use. The protein concentrations of the extracts were determined according to the Bradford

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method 23 using the Bio-Rad Protein Assay. Bovine serum albumin (BSA) at a concentration

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of 1 mg/mL was used as standard.

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Dot-Blot analysis

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The abundance of 18 proteins (including intact molecules and their fragments)

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corresponding to five different biological functions (Table 1): muscle fibre structure (Actin,

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MyBP-H, CapZ-β and MyLC-1F); metabolism (ENO3, LDHB and MDH1); proteolysis (µ-

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calpain); oxidative resistance (DJ-1, Prdx6 and SOD1); and Heat shock proteins (αB-

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crystallin, Hsp20, 27, 40, 70-1A/B, 70-8 and 70-Grp75) were determined using Dot-Blot

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technique

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coefficient of variation inter and intra assay (10%). Optimal dilution ratios of the antibodies

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were determined, using the conditions indicated by the supplier and adapted to bovine muscle

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samples(Table 1). For that, western blots were used in order to check the specificity of all the

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antibodies. An antibody was considered specific against the studied protein when only one

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. Compared to Western Blot, Dot-Blot is a fast technique, but with a similar


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band at the expected molecular weight was detected by western blot. Western blots with all

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primary antibodies show that all the antibodies used bound specifically to the bovine protein

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with the expected theoretical molecular weight.

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Conditions retained and suppliers for all primary antibodies dilutions are summarized in

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Table 1. Protein extracts (15 µg) of each of the 21 muscle samples were spotted (4

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replications per muscle sample) on a nitrocellulose membrane with the Minifold I Dot-Blot

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apparatus from Schleicher & Schuell Biosciences (Germany) in a random order on the 96-

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spots membrane. In addition, a mixed standard sample (15 µg) was deposited for data

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normalization as reported by Guillemin et al. 15. The Dot-Blot membrane was air-dried for 5

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minutes, and blocked in 10% milk blocking buffer at 37°C for 20 minutes and then hybridized

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and incubated with the specific primary antibody of each protein (Table 1). Infrared

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fluorescence detection was then used for quantification of the relative protein abundances.

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Subsequently, the membranes were scanned by the Odyssey scanner (LI-COR Biosciences) at

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800 nm. The relative protein abundances for each sample were given in arbitrary units.

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Electrophoresis and quantification of Myosin Heavy Chain (MyHC) isoforms

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Three other biomarkers corresponded to MyHC and were determined according to Picard

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et al. 24 using an adequate SDS-PAGE and expressed in percentage. Briefly, 100 mg of frozen

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muscle were ground in 5mL of extraction buffer solution containing 0.5 M NaCl, 20 mM Na

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Pyrophosphate, 50 mM Tris, 1 mM EDTA and 1 mM Dithiothreitol. After 10 min at 4°C on

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ice, the sample was centrifuged for 5 min at 5000g. Following centrifugation, the supernatant

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was diluted 1:1 (v/w) with glycerol at 87% and stored at –20°C until use. The samples were

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then mixed with an equal volume of loading buffer containing 4% SDS (w/v), 125 mM Tris,

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pH 6.8, 20% glycerol (v/v), 10% β-mercaptoethanol (v/v) and 0.02% pyronin Y (w/v). The

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proteins were separated using 9.2% polyacrylamide gels. After staining, the gels were

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scanned and the proportions of the different MyHCs bands were quantified by densitometry

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with ImageQuant Software 5500 (Amersham Biosciences/GE Healthcare). The bands

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quantification revealed the existence of MyHC-IIb isoforms in only some animals (4 animals

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of 21). Consequently MyHC-IIb percentages were totalled with those of MyHC-IIx creating a

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new variable “MyHC-IIx+b” (fast glycolytic fibres).

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Statistical analysis For descriptive statistics, raw data were used. For statistical analyses all data were

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standardized for replicate using the Proc Standard of SAS 9.2 to obtain Z-scores. A Z-score 5 ACS Paragon Plus Environment


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represents the number of standard deviations each observation is relative to the mean of the

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corresponding animal: =

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µ

where x is the raw value, µ is the mean of the population and

σ is the standard deviation of the same population.

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The PROC CORR of SAS was used to determine the Pearson's correlation coefficients

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between the 21 biomarkers with pH and CIE-L*a*b* colour coordinates. Correlation values

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were considered significant at P < 0.05.

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For each pH value and each colour coordinate, a Principal Component Analysis (PCA) was

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carried out using PROC PRINCOMP of SAS, using only the correlated biomarkers. They

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aimed to illustrate visually the correlated biomarkers with pH and colour coordinates. The

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overall Kaiser's Measure of Sampling Adequacies of the performed PCA’s were > 0.68 for pH

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parameters and > 0.80 for L* a* b* colour coordinates.

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Multiple regression analyses were performed using PROC REG of SAS to create best

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models (maximal adjusted r2) for pH and colour coordinates (as dependent variables) using

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the 21 protein biomarkers (as independent variables). Partial R-squares and significance of

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each retained variable are given for the models. Regression analyses were further conducted

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on L*a*b* colour coordinates to study specifically the relationships with pH values and

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muscle fibre type composition.

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The absence of collinearity was systematically verified for each model, by producing

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condition indices and variance proportions using the COLLIN procedure of SAS. Variables

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were identified as collinear if they possessed both a high condition index > 10 and a

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proportion of variation > 0.5 for two or more traits. RESULTS

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pH parameters, colour traits and biomarkers abundances

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Average means of pH and colour parameters are shown in Fig. 1. pH measured 45 min p.m

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was 6.87 (± 0.10) and declined to 6.22 (± 0.20) 3 h p-m reaching 5.57 (± 0.08) 30 h p.m. (Fig.

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1a). Mean colour coordinates were 36.9 (± 3.32) for lightness (L*), 12.40 (± 1.74) for redness

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(a*) and 15.81 (± 2.65) for yellowness (b*) (Fig. 1b). Relative abundances of biomarkers

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expressed in arbitrary units or in percentage (MyHC) are summarised in Table 2. The LT

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muscle was characterized by 19.2 % (± 3.6) of type I fibres, 3.9 % (± 4.3) of IIa, and 56.9 %

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(± 4.0) of IIx/b. 6 ACS Paragon Plus Environment

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Relationships between biomarkers and pH parameters

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pHu was correlated with pH45min (r = 0.47; P < 0.05) and pH3h (r = 0.59; P < 0.01). pH45min

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was correlated with pH3h (r = 0.47; P < 0.05). pH was further correlated with 11 biomarkers

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(Fig. 2a). Prdx6 was negatively correlated with pH45min (r = –0.49, P < 0.05), pH3h (r = –0.47,

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P < 0.05) and pHu (r = –0.66, P < 0.01). Hsp70-Grp75 and DJ-1 were negatively correlated

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with both pH3h (r = –0.45 and –0.56, P < 0.05; respectively) and pHu (r = –0.45 and –0.43, P

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< 0.05; respectively). Actin was negatively correlated with pH45min and pHu (r = –0.40, P

Understanding Early Post-Mortem Biochemical Processes Underlying

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